FGFR3 Heterodimerization in Achondroplasia, the Most Common Form of Human Dwarfism

Abstract

The G380R mutation in the transmembrane domain of fibroblast growth factor receptor 3 (FGFR3) causes achondroplasia, the most common form of human dwarfism. Achondroplasia is a heterozygous disorder, and thus the affected individuals express both wild-type and mutant FGFR3. Yet heterodimerization in achondroplasia has not been characterized thus far. To investigate the formation of FGFR3 heterodimers in cellular membranes, we designed an FGFR3 construct that lacks the kinase domain, and we monitored the formation of inactive heterodimers between this construct and wild-type and mutant FGFR3. The formation of the inactive heterodimers depleted the pool of full-length receptors capable of forming active homodimers and ultimately reduced their phosphorylation. By analyzing the effect of the truncated FGFR3 on full-length receptor phosphorylation, we demonstrated that FGFR3 WT/G380R heterodimers form with lower probability than wild-type FGFR3 homodimers at low ligand concentration. These results further our knowledge of FGFR3-associated bone disorders.The G380R mutation in fibroblast growth factor receptor 3 (FGFR3) transmembrane (TM) 2 domain has been linked to achondroplasia, the most common form of human dwarfism (1). Achondroplasia, characterized by short stature, is a common (1 in 15,000 live births) autosomal dominant disorder that interferes with the maturation of the cartilage growth plate of long bones (2-4). The affected protein, FGFR3, is a receptor tyrosine kinase. Its activation, manifested in the autophosphorylation of its kinase domain, involves lateral dimerization and binding of FGF ligands and heparan sulfate (5, 6). Ligand binding increases receptor activation by stabilizing the dimers and perhaps altering the dimer structure (7-11).In cellular studies, the G380R mutation increases FGFR3 phosphorylation in the absence of ligand and at low ligand concentration (12-15). The increase, which is the likely cause for pathogenesis, has been linked to an increase in the phosphorylation of the unliganded dimer (13). However, FGFR3 dimerization does not appear affected by the G380R mutation, as both the wild-type FGFR3 and the G380R mutant show very similar cross-linking and dimerization propensities (13).Achondroplasia is a heterozygous disorder, and thus the affected individuals express both wild-type and mutant FGFR3. In this paper, we address the question whether mutant FGFR3 receptors heterodimerize with the wild type. Currently, the answer to this question is unknown. Most studies of FGFR3 activation thus far have focused on FGFR3 homodimers, because the detection of heterodimers is challenging. When both wild-type and mutant FGFR3 molecules are present in cells, three different dimeric species can co-exist as follows: 1) wild-type homodimers, 2) mutant homodimers, and 3) wildtype/mutant heterodimers (16). These dimers will be indistinguishable in many experiments, and their abundance will depend on the respective expression levels and on the dimerization propensities, which are unknown. Furthermore,...

Effect of the achondroplasia mutation on FGFR3 dimerization and FGFR3 structural response to fgf1 and fgf2: A quantitative FRET study in osmotically derived plasma membrane vesicles

“…This finding is consistent with previous findings that (i) the achondroplasia mutation increases FGFR3 phosphorylation in the absence of ligand [11,12,23], and (ii) the effect of the mutation on phosphorylation gradually disappears as the ligand is titrated [12,52]. …”

Achondroplasia: Development, pathogenesis, and therapy

“…This mutation is localized in the transmembrane domain of the receptor distal to the recurrent Ach mutation (p.Gly380Arg). Differences in phenotypes (craniosynostoses vs chondrodysplasia) of the p.Ala391Glu and p.Gly380Arg mutations may be attributed to relative increased formation of FGFR3 heterodimers with the p.Ala391Glu mutation (He et al, 2011). …”

A New Method to Study Heterodimerization of Membrane Proteins and Its Application to Fibroblast Growth Factor Receptors

“…For instance, the ErbB2⅐ErbB3 heterodimer is known as the most biologically active and the most pro-tumorigenic of all ErbB homodimers and heterodimers (4, 15). However, our understanding of RTK heterodimerization is only rudimentary, in part because of a paucity of methods that provide quantitative information about heterodimer formation (18,20,21). Indeed, prior work has relied primarily on qualitative methods such as immunoprecipitation.…”

“…Thus, this FRET method can have broad utility in membrane protein research, because it can be used to study the heterodimerization propensity of any two membrane proteins. (20,29,60,61), but the thermodynamics of the association process have not been quantified. Furthermore, FGFR heterodimerization in the absence of ligand has never been investigated.…”